Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.
Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.
Usmanova
A
A
Centre de Recherches de Biochimie Macromoléculaire (CNRS), U249 (INSERM), Université de Montpellier I, France.
Astier
C
C
Lebart
M C
MC
Kwiatek
O
O
Papa
I
I
Boyer
M
M
Roustan
C
C
Benyamin
Y
Y
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001492:Bass; D051347:CapZ Actin Capping Protein; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008274:Magnesium / Q000494:pharmacology; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000199:Actins; D051347:CapZ Actin Capping Protein; D008840:Microfilament Proteins; D009124:Muscle Proteins; D003850:Deoxyribonuclease I; D008274:Magnesium